1. Field of the Invention
The present invention relates to a flow cytometry apparatus, and more particularly, concerns a flow cytometry apparatus for determining characteristics of cells or the like, which includes improved optics for focusing the light beam on the moving cells.
2. Description of the Prior Art
Flow cytometry apparatuses rely upon the flow of cells or other particles in a liquid flow stream in order to determine one or more characteristics of the cells under investigation. For example, a liquid sample containing cells is directed through the flow cytometry apparatus in a fast moving liquid stream so that each cell passes, substantially one at a time, through a sensing region. Changes in electrical impedance as each cell passes through the sensing region have been associated with the determination of cell volume. Similarly, if an incident beam of light is directed at the sensing region, the passing cells scatter such light as they pass therethrough. This scattered light has served as functions of cell shape, index of refraction, opacity, roughness and the like. Further, fluorescence emitted by tagged cells which have been excited as a result of passing through the excitation energy of the incident light beam is detectable for the identification of specifically marked cells. Not only is cell analysis performed on the flow cytometry apparatuses, but sorting of cells is also achieve. Lasers have been used as the source of the incident beam of illumination in flow cytometry apparatuses, as well as sources of incoherent light, such as mercury arc lamps.
In laser excited flow cytometry, in particular, a tightly focused laser beam is typically brought into coincidence with the cells or the like which are to be analyzed in conjunction with the light beam. Thus, the light beam allows the analysis to be conducted as a result of light scattered by the cells or fluorescence emitted thereby. As the cells travel in a normally vertical trajectory in their liquid flow path from the nozzle tip to the cell collector, they pass through the focused laser beam which normally travels on a substantially horizontal trajectory. The optical system, frequently multichannel, for measuring fluorescence or light scatter typically has a viewing axis which is mutually normal to both the liquid flow stream and the laser beam. Whenever the laser beam intercepts a cell, an optical pulse is generated, the intensity and wavelength profile of which characterizes the cell. Optical pulses are ultimately converted to digits and processed by a computer according to preselected operator functions. Output data is typically presented to the operator in conjunction with this digital information. Since it is desirable that the pulses provide intensity information about the cell rather than the laser beam, it is further desirable that the vertical dimension of the focused laser spot be reduced so as to be smaller than the typical cells under investigation.
Moreover, the electrical measuring system for analyzing the pulses is sometimes concerned with only the height of the pulses rather than the area. In this event, reduction of the vertical focal waist of the laser beam to that approaching the cell diameter causes an increase in the gathered signal in direct proportion thereto. On the other hand, reduction of the horizontal focal waist of the laser beam to less than the cell diameter usually leads to deleterious results by magnifying the uncertainty in the precise horizontal position of the liquid flow stream. In this event, poorer resolution results, which in this field of technology, is referred to as increased coefficient of variation. Accordingly, an improved illuminating system desirably would include a focal region with the vertical beam waist substantially smaller than the horizontal beam waist. Such asymmetric beam shaping is not uncommon in laser flow cytometry.
For instance, it is well understood that single mode continuous wave lasers of the type used in flow cytometry apparatuses have beam intensity profile functions which are Gaussian and that the following equation expresses the beam waist when focused with a lens: EQU .delta.=(4.lambda.f)/(.pi.w)
Where,
.lambda. is laser wavelength; PA1 f is lens focal length; PA1 w is width of the unfocused laser beam; and PA1 .delta. is minimum focal waist as limited by diffraction. PA1 (w and .delta. are usually dimensioned to the 1/.epsilon..sup.2 intensity points.
From the above equation it is evident that to reduce the beam waist, either the lens focal length (f) must be decreased or the unfocused laser beam width (w) must be increased or expanded. Of course, either approach is successful only so long as the angles are small enough so that geometric abberations are not a factor.
Currently, the most common way of producing a focused beam waist which is smaller in the vertical plane than the horizontal, relies upon cylindrical lenses. Typically, an elliptical beam shape can be created in which the vertical beam width is greater than the horizontal beam width, and then focusing can be achieved with a spherical lens having a specific focal length. Alternatively, it has been known to rely on a circular incident beam focused on the cell by asymmetric, usually cylindrical, focusing optics. The same Gaussian relationship pertains to this type of focusing optics. Any number of variations of these techniques may be used to create an asymmetric spot on the flowing cells in the liquid flow stream.
All of the known prior art systems have a chromatic problem when more than one laser is employed simultaneously as the source of excitation light, and when relying on one of the above-described cylindrical lens focusing approaches. Each of the cylindrical lenses focuses the different wavelength lasers at concomitantly different distances from the lens. Thus, where the flowing liquid stream of cells would intersect the first laser in a tightly focused zone, the cell would, a few microseconds later, intersect the second laser beam on a larger substantially defocused zone with a corresponding degradation in performance. If cylindrical lenses are used, rectification of this problem requires two or three complicated element lenses carefully designed with different glasses of appropriately varying dispersions. Usually, such combinations may correct chromatic abberation adequately over only a part of the required wavelength region. Furthermore, the axes of the individual cylindrical elements need to be carefully controlled. Even if the cylindrical lenses are carefully configured, they may contribute optical abberations which have to be kept within the diffraction limit at the cell space in order to produce a beam shape for optimum performance.
As a result of the foregoing deficiencies, there is clearly a need for improvement in light beam shaping for flow cytometry apparatuses in order to produce a focal region with the vertical beam waist substantially smaller than the horizontal beam waist. It is to such an improvement that the present invention is directed.